U.S. patent number 4,748,002 [Application Number 06/866,956] was granted by the patent office on 1988-05-31 for semi-automatic, solid-phase peptide multi-synthesizer and process for the production of synthetic peptides by the use of the multi-synthesizer.
This patent grant is currently assigned to Centre National de la Recherche Scientifique (CNRS). Invention is credited to Jean-Paul Briand, Jean Neimark.
United States Patent |
4,748,002 |
Neimark , et al. |
May 31, 1988 |
Semi-automatic, solid-phase peptide multi-synthesizer and process
for the production of synthetic peptides by the use of the
multi-synthesizer
Abstract
A semi-automatic, solid phase peptide multi-synthesizer is
characterized in that it is principally constituted by chemical
reactors (2), connected to volumetric solvent proportioners (1)
disposed above the said chemical reactors (2) and below a solvent
selector (4) supplied with solvent by solvent containers (6) under
pressure of nitrogen distribution cylinders (3), as well as a
center for adjustment and regulation of very low pressure nitrogen
(5), are sequentially controlled by a programmable automation (7)
or a microcomputer assuring the carrying out of the synthesis
technique. The maximum number of volumetric solvent proportioner
(1)--chemical reactor (2) assemblies is equal to the maximum number
of peptides which may be produced simultaneously.
Inventors: |
Neimark; Jean (Strasbourg,
FR), Briand; Jean-Paul (Strasbourg, FR) |
Assignee: |
Centre National de la Recherche
Scientifique (CNRS) (Paris, FR)
|
Family
ID: |
9319872 |
Appl.
No.: |
06/866,956 |
Filed: |
May 27, 1986 |
Foreign Application Priority Data
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Jun 3, 1985 [FR] |
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85 08438 |
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Current U.S.
Class: |
422/116;
525/54.11; 530/334; 422/131; 530/333 |
Current CPC
Class: |
C07K
1/045 (20130101); B01J 19/0046 (20130101); C40B
60/14 (20130101); B01J 2219/00286 (20130101); B01J
2219/0059 (20130101); B01J 2219/00596 (20130101); B01J
2219/00493 (20130101); B01J 2219/00423 (20130101); B01J
2219/00725 (20130101); B01J 2219/00389 (20130101); B01J
2219/005 (20130101); C40B 40/10 (20130101) |
Current International
Class: |
B01J
19/00 (20060101); C07K 1/00 (20060101); C07K
1/04 (20060101); C07K 001/04 (); C07K 001/06 ();
C07K 001/10 (); B01J 008/02 () |
Field of
Search: |
;530/333,334
;525/54.1,54.11 ;422/116,131 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0042792 |
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Dec 1981 |
|
EP |
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0130739 |
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Jan 1985 |
|
EP |
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2554820 |
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May 1985 |
|
FR |
|
Other References
"An Automatic Apparatus for the Synthesis of Peptides Using Resin
Coated Glass Beads in the Form of a Packed Bed", Journal of
Chromatographic Science, vol. 10, Jun. 1972, by R. P. W. Scott et
al., pp. 384-391..
|
Primary Examiner: Phillips; Delbert R.
Assistant Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Young & Thompson
Claims
What is claimed is:
1. In a semi-automatic, solid phase peptide multi-synthesizer of
the type comprising a hydraulic circuit having: a plurality of
chemical reactors; a corresponding plurality of volumetric solvent
proportioners, each of said plurality of chemical reactors being
disposed downstream of a corresponding said volumetric solvent
proportioner; a plurality of solvent containers disposed upstream
of said plurality of volumetric solvent proportioners; distribution
means disposed intermediate said solvent containers and said
solvent proportioners, for directing a desired solvent from its
corresponding said solvent container to any selected one of said
plurality of volumetric solvent proportioners; and a programmable
microcomputer implementing a predetermined sequence in which fluids
are supplied to and evacuated from said plurality of chemical
reactors and said corresponding plurality of volumetric solvent
proportioners; the improvement in which said distribution means
comprises a first cylindrical element traversed by an axial opening
forming a distribution channel having inlet and outlet ends, a
plurality of radial openings communicating with said distribution
channel and offset axially of said cylindrical element, said
cylindrical element being disposed substantially vertically within
said multi-synthesizer such that said inlet end underlies said
outlet end, each said radial opening being connected to a said
volumetric solvent proportioner and said inlet end of said
distribution channel receiving solvents from said solvent
containers.
2. Peptide multi-synthesizer according to claim 1, further
comprising a frame having a lower portion enclosing said plurality
of solvent containers, a central portion comprising a work table
above which are disposed said plurality of chemical reactors, and
an upper portion enclosing said volumetric solvent proportioners,
said distribution means and said programmable microcomputer.
3. Peptide multi-synthesizer according to claim 1, wherein said
plurality of chemical reactors and said corresponding plurality of
volumetric solvent proportioners are mounted in parallel by means
of tubes interconnected by unifying connectors, said tubes
comprising valves for isolating any selected one of said plurality
of volumetric solvent proportioners and its corresponding said
chemical reactor, said tubes and said unifying connectors being
formed from polytetrafluoroethylene (PTFE) or fluorinated
ethylenepropylene copolymer (FEP).
4. Peptide multi-synthesizer according to claim 1, wherein each of
said plurality of volumetric solvent proportioners comprises a
principal cylinder for introduction of solvents under pressure of
nitrogen and a measuring column for determining exact volume of
solvent to be injected into a corresponding said chemical reactor,
said principal cylinder comprising a column having an upper opening
forming a principal inlet for solvents under pressure of nitrogen,
said column projecting upwardly and outwardly from said principal
cylinder, said column having a lower end opening onto a disk
connected to said column, said principal cylinder comprising a
lower outlet communicable with its corresponding said chemical
reactor, said measuring column having an upper inlet for reception
of high pressure nitrogen and evacuation of air, evacuation means
being connected to said upper inlet of said measuring column, said
evacuation means comprising an activated charcoal filter and an
extractor, said measuring column being in continuous communication
with said principal column.
5. Peptide multi-synthesizer according to claim 1, wherein each of
said plurality of chemical reactors comprises a first upper opening
comprising an injection device for pressurized solvent, said first
upper opening being communicable with its corresponding said
volumetric solvent proportioner, a second upper opening for
introduction of amino acids and resin and for withdrawing samples
of peptide-resin couples, a third upper opening for evacuation of
said reactor, evacuation means being connected to said third upper
opening and comprising an activated charcoal filter and an
extractor for evacuation of air or residual nitrogen contained in
said reactor, said second and third upper openings flanking said
first upper opening, and a lower opening for evacuation of solvents
and admission of low pressure nitrogen for agitation by bubbling,
all said openaings of said reactor comprising control valves
permitting successive or simultaneous control of admission and
evacuation of fluids from said reactor.
6. Peptide multi-synthesizer according to claim 5, wherein said
injection device of said first upper opening of each said reactor
comprises an upper coupling connector comprising a hollow cylinder
containing an open, ribbed cylinder of slightly lesser diameter,
said ribbed cylinder having a lower base comprising an enlarged
frusto-conical portion permitting radial and laminar distribution
of liquid flux injected into said reactor.
7. Peptide multi-synthesizer according to claim 6, wherein said
enlarged frustoconical portion of said ribbed cylinder comprised by
said injection device of said first upper opening of said chemical
reactor defines a conical angle comprised between 50.degree. and
70.degree. and has a height about 10 mm less than said first upper
opening of said reactor, whereby liquid injected through said
injection device contacts interior surfaces of said chemical
reactor and said second and third upper openings thereof.
8. Peptide multi-synthesizer according to claim 1, wherein said
distribution means further comprises a second cylindrical element
disposed vertically within said multi-synthesizer intermediate said
solvent containers and said first cylindrical element, said second
cylindrical element being traversed by an axial opening forming a
distribution channel having first and second ends, a plurality of
radial openings communicating with said distribution channel and
each disposed on a different level of said second cylindrical
element according to a helical repeating interval, each said radial
opening of said second cylindrical element receiving solvent from a
said solvent container, said upper end of said distribution channel
of said first cylindrical element communicating with said inlet end
of said distribution channel fo said first cylindrical element,
said lower end of said distribution channel of said second
cylindrical element communicating with a waste recovery device.
9. In a semi-automatic, solid phase peptide multi-synthesizer of
the type comprising a hydraulic circuit having: a plurality of
chemical reactors; a corresponding plurality of volumetric solvent
proportioners, each of said plurality of chemical reactors being
disposed downstream of a corresponding said volumetric solvent
proportioner; a plurality of solvent containers disposed upstream
of said plurality of volumetric solvent proportioners; distribution
means disposed intermediate said solvent containers and said
solvent proportioners, for directing a desired solvent from its
corresponding said solvent container to any selected one of said
plurality of volumetric solvent proportioners; and a programmable
microcomputer implementing a predetermined sequence in which fluids
are supplied to and evacuated from said plurality of chemical
reactors and said corresponding plurality of volumetric solvent
proportioners; the improvement in which each said volumetric
solvent proportioner comprises a measuring column associated with a
strip of optoelectronic level detectors, said computer being
programmed to halt supply of solvent to each said volumetric
solvent proportioner responsive to detection by its corresponding
said detectors of solvent in said measuring column having attained
a predetermined level.
Description
The present invention, realized in laboratory LP 6201 of the
Institute of Molecular and Cellular Biology of the NATIONAL CENTER
FOR SCIENTIFIC RESEARCH (NCSR), concerns the chemical synthesis of
peptides, and has as an object a peptide multi-synthesizer
comprising means for assuring the simultaneous synthesis of a
variable number of peptides in differing quantities and having
identical or different sequences.
At present, none of the existing apparatus permits the simultaneous
synthesis of more than two peptides with different sequences. The
synthesizers which exist are either mono-synthesizers, or
bi-synthesizers, and are manual, semi-automatic or automatic. The
mono-synthesizers are capable of synthesizing only a single peptide
at a time, and the bi-synthesizers, which use two reactors, can
synthesize simultaneously only two different peptides. But, the
synthesis of a peptide of 20 to 30 amino acids with verification of
each amino acid linkage generally requires about ten days. With
such apparatus, it is reasonable to expect a production of only
four synthetic peptides per month. Moreover, it must be noted that
each of the automatic apparatus on the market at present does not
effect control tests at the essential stages of the synthesis.
The present invention has as an obect to permit simultaneously the
synthesis of a variable number of peptides with identical or
different sequences according to a common synthesis technique.
Specifically, it has as an object a semi-automatic, solid-phase
peptide multi-synthesizer, characterized in that it is principally
constituted by chemical reactors connected to volumetric solvent
proportioners disposed above the said chemical reactors and below a
solvent selector supplied with solvent by solvent containers under
pressure of nitrogen, by distribution cylinders, as well as by a
center for adjustment and regulation of very low pressure nitrogen,
these means being sequentially controlled by a programmable
automaton or a microcomputer assuring the carrying out of the
synthesis technique, and the maximum number of volumetric solvent
proportioner-chemical reactor assemblies as being equal to the
maximum number of peptides which may be simultaneously
synthesized.
In the past years, the chemical synthesis of peptides has advanced
considerably. Specifically, it has been established that numerous
peptides play a fundamental role in the communication between
neurons. The use of synthetic peptides has thus permitted rapid
progress in the understanding of the mechanism of action of
neuropeptides and in their therapeutic utilization.
But the use of synthetic peptides has similarly taken a dominant
place in several other fields. In immunology, this technology
constitutes a future path toward the obtention of synthetic
vaccines. There are synthesized, in this case, peptides
corresponding to the antigenic regions of viral proteins. Recent
results show that the coupled peptides, injected in an animal, may
protect it from viral infection. Use of such synthetic vaccines
will permit avoiding the inherent problems in the production of
known vaccines, particularly the accidental persistence of
pathogenic agents in the inactivated virus preparations, and the
injection of viral nucleic acid that is always present in these
preparations.
In molecular biology, thanks to the developments of genetic
engineering, it is possible to arrange relatively easily the coding
gene sequence for functional proteins or structure proteins. Thus,
these nucleotidic sequences are decoded and the amino acid
sequences of these proteins are obtained without having had either
to isolate or to purify them. It is nevertheless necessary to know
if these proteins are actually present in the cellular system under
study. It is at this stage that the use of synthetic peptides
intervenes. The peptidic fragments previously mentioned as being
probable antigenic determinants of the protein under study are
synthesized, and they are coupled to a carrier protein before being
injected into the animal. Given that the anti-peptide antibodies
are capable, in most cases, of identifying the native protein, it
can therefore be known if the protein is present or not in the
cell. With the aid of immunoadsorption columns, one may thus
envision purifying the protein in question starting from a crude
cellular extract. It is similarly possible to observe, for example,
the fate of various regions of a protein when this latter undergoes
a process of maturation.
Various synthesis techniques are known. The technique most used
currently is that developed by Merrifield. His principle is as
follows:
The amino acids are sequentially added to the growing chain, the
C-terminal extremity of which is connected to an insoluble solid
support. All of the reactions take place in the same vessel and the
excess reagents are eliminated by simple filtration and washings.
The sequence of operations is effected in the following manner:
##STR1##
The solid support is a polystyrene resin polymerized with 1%
divinylbenzene and comprising a great number of groups capable of
reacting with the --COOH group of the amino acids. During the
condensation step, the N.alpha. amino group of the added amino acid
must be protected: the protecting group most used is
t-butyloxycarbonyl (BOC). The secondary functional group is
similarly protected during the entire synthesis. They are unblocked
only at the time of the liberation of the peptide from the resin,
by hydrofluoric acid.
The t-butyloxycarbonyl group is eliminated by acidolysis in a
trifluoroacetic acid-dichloromethane system containing an
anti-oxidizing agent. The amino group obtained in the form of a
salt is then neutralized in the presence of a base such as N,N
diisopropylethylamine. These two principal steps are separated by
several washing steps. Then the deprotection reaction is verified
by a test with ninhydrin.
The condensation agent used is N,N'dicyclohexylcarbodiimide which
activates the carboxyl group of the t-butyloxycarbonyl
protector-amino acid grouping to be added, in O-acyl urea. This
intermediate may also be rapidly isomerized in inactive N-acyl
urea.
The reaction conditions are as follows:
*t-butyloxycarbonyl-amino-acid group: molar excess.times.3
*N,N'dicyclohexylcarbodiimide of the dichloromethane for 1 to 2
hours.
Variations of the coupling reaction consist in adding the amino
acid in a pre-activated form. The two most used forms are the
activated esters or the symmetric anhydrides of amino acids.
Whatever may be the method of coupling employed, the condensation
reaction is verified by the test with ninhydrin. If the coupling
reaction is not complete, a double condensation is effected, that
is to say that the neutralization step is repeated. Specifically,
it is possible that a slight percentage of peptide-resin remains in
the salt form. The t-butyloxycarbonyl-amino-acid group and the
N,N'dicyclohexylcarbodiimide are the added in 1.5 molar excess for
30 min. to 1 hour.
If the ninhydric test still remains slightly positive, the
remaining free N amino groups are blocked, by an acetylation
reaction, which could give rise to additional reactions at the time
of subsequent condensations.
______________________________________ *acetic anhydric molar
excess .times. 10 *N,N diisopropylethylamine molar excess .times.
11 ______________________________________
in dichloromethane for 10 min.
There exists a variant of the Merrifield technique proposed by
Sheppard. The general principle is the same, but the nature of the
protecting groups, the deprotecting solvents and if desired the
resin, is changed.
Merrifield has elaborated this technique so as to permit its
automation. Indeed, the quality of the final product, that is to
say that of the pepetide obtained with an automatic, semi-automatic
or manual synthesis apparatus is the same whatever may be the
conception of the said apparatus, but the use of a manual synthesis
apparatus requires attention such that the permanent presence of
the operator is essential. Nevertheless, the use of an entirely
automatic apparatus does not permit immediate quality control at
each fundamental step of the synthesis and this is why the present
invention concerns a semi-automatic peptide multi-synthesizer
seeking the obtention of a final product of very high purity.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be best understood thanks to the following
description, which relates to preferred embodiments, given by way
of non-limiting examples and explained with reference to the
accompanying schematic drawings, in which:
FIG. 1 is a perspective view of the peptide multi-synthesizer
according the the invention;
FIG. 2 is a sectional view of a volumetric proportioner;
FIG. 3 is a sectional view of a chemical reactor;
FIG. 4 is a sectional view of an injection device;
FIG. 5 is a perspective view of a distribution cylinder;
FIG. 6 is a perspective view of the solvent selector;
FIG. 7 is a view of the principal connection diagram for a
volumetric solvent proportioner-chemical reactor, and
FIG. 8 is a view of the fluidic and pneumatic principle functioning
diagram .
DETAILED DESCRIPTION OF THE INVENTION
According to the invention, the peptide multi-synthesizer is
principally constituted by chemical reactors 2 connected to
volumetric solvent proportioners 1 disposed above the said chemical
reactors 2 and below a solvent selector 4 supplied by solvent
containers 6 under pressure of nitrogen, by distribution cylinders
3, as well as by a center for adjustment and regulation of very low
pressure nitrogen 5, these means being sequentially controlled by a
programmable automaton 7 or a microcomputer assuring the carrying
out of the synthesis technique, and the maximum number of
volumetric solvent proportioner 1-chemical rector 2 assemblies
being equal to the maximum number of peptides which may be produced
simultaneously.
As shown in FIG. 1, the peptide multi-synthesizer has a metallic
frame 8 the lower part of which encloses the center 5 for
adjustment and regulation of very low pressure nitrogen as well as
the containers 6 of solvent under pressure of nitrogen, the central
portion of which is constituted by a work table 9 behind which is
found the region of the output valves of the chemical reactors 2
and above which is situated the region of the chemical reactors 2,
and the upper part of which encloses the volumetric solvent
proportioners 1, behind which is found the various distribution
cylinders 3, the solvent selector 4, as well as the programmable
automaton 7 or the microcomputer.
The front surface of the upper part has various dials, one dial in
particular of which illustrating the synoptic diagram of the
fluidic and pneumatic operation of the device, a dial for control
and programming of the automaton as well as a dial for control of
irregulatities. The solvent containers 6 are disposed on the base
of the metallic frame 8. All of the other elements, particularly
the center 5 for adjustment and regulation, the volumetric solvent
proportioners 1, the chemical reactors 2, the distribution
cylinders 3, the solvent selector 4 and the programmable automaton
7 or the microcomputer are fixed to the different walls and the
different uprights of the frame 8 in a known manner, with the aid
of fastening and gripping collars, fastening braces, assembly
plates, gripping plates and rods, fastening staples, as well as
various types of screws and bolts. The supply of nitrogen will be
assured, as shown in FIGS. 7 and 8, by a nitrogen container 48
through an expansion device 47 which permits lowering the pressure
delivered by the nitrogen container from 200 bar to 15 bar for
suppying through a control valve 51 two expansion valve 46-humidity
filter 45 assemblies provided respectively for supplying the center
5 for adjustment and regulation. The nitrogen container 48 is
disposed near the apparatus and connected to one of the
distribution cylinders 3. The volumetric solvent proportioner
1-chemical reactor 2 assemblies are mounted in parallel by means of
tubes 10 interconnected by unifying junctions 11, and in which are
injected solvents or nitrogen, valves 12 being inserted in the
tubes 10, by junctions 11' between valves 12 and tubes 10, the
unifying junctions 11 and the valve-tube junctions 11' as well as
the tubes 10 being realized in polytetrafluoroethylene (PTFE) or in
fluorinated ethylenepropylene copolymer (FEP).
All the junctions are of a known type, perferably from the
Bohlender society. The number of volumetric solvent proportioner
1-chemical reactor 2 assemblies may vary from one apparatus to
other. In the embodiment described there will advantageously be
six, that is to say six volumetric solvent proportioners 1 and six
chemical reactors 2. It will therefore be possible to produce
simultaneously six peptides of identical or different sequences and
in different quantities. These latter may vary from about 100
milligrams to several grams. Each peptide will be realized in a
volumetric solvent proportioner 1-chemical reactor 2 assembly, and
this in a totally independent manner from the production of the
other peptides.
As shown in FIG. 2, each volumetric solvent proportioner 1 is
constituted by two cylinders, namely a principal cylinder 13 for
introduction of the various solvents under pressure of nitrogen and
a measuring column 14 for determination of the exact volume of
solvent to be injected into the chemical reactor 2. The principal
cylinder 13 is traversed by a column 15 the upper opening 16 of
which is the principal inlet for the various solvents under
pressure of nitrogen and projects with respect to the upper
extremity of the principal cylinder 13, and the lower extremity 17
of which opens onto a disk 18 connected to the column 15 by two
attachments 19 and 20, the lower extremity of the principal
cylinder 13 being provided with an outlet opening 21 connected to a
column 22 situated in the extension of the principal cylinder 13
for injection of a solvent under pressure of nitrogen into the
chemical reactor 2. The measuring column 14 has, on the one hand,
at its upper extremity an opening 23 for admittance of high
pressure nitrogen and escape of air, connected, as shown in FIG. 8,
to a device for evacuation under slight underpressure constituted
by an activated charcoal filter 49 and an extractor 50, and, on the
other hand, near this said upper extremity, a connection conduit 24
to the principal cylinder 13, its lower extremity being connected
to the column 22. The measuring column 14 is associated with a
strip 25 of optoelectronic detectors determining the various
volumes of solvent. These optoelectronic detectors are
advantageously positioned so as to select seven volumns of solvent,
namely 5 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml and 70 ml. The
preselection of the volume is realized by programming as a function
of the quantity of product to be produced by the chemical reactor 2
which is associated with it. The volumetric solvent proportioners 1
thus permit simultaneously injecting various solvents in different
quantities into the chemical reactors 2.
According to FIG. 3, each chemical reactor 2 has four openings,
namely an opening 26 situated at the top of the chemical reactor 2,
containing an injection device 27 for solvent under pressure, as
well as nitrogen under pressure at the time of evacuating the
chemical reactor of its liquid, and connected by a T-shaped
valves-junction assembly to the column 22 of the volumetric
proportioner 1, an opening 28 for introduction of amino acids and
of the resin similarly permitting the sample retention of the
resin-peptide couples for the control, at the essential stages of
the synthesis, and opening 29 connected, as shown in FIG. 8, to an
evacuation device under weak underpressure constituted by an
activated charcoal filter 49 and an extractor 50 for the evacuation
of air or residual nitrogen retained in the chemical reactor 2 at
the time of its refilling, the two openings 28 and 29 being
situated on either side of the opening 26, and an opening 30 for
evacuation of the solvents and admission of nitrogen under very low
pressure for agitation by bubbling, and situated at the lower
extremity of the chemical reactor 2. The experimenter places the
resin in the interior of the various chemical reactors 2, resin to
which is coupled the growing amino-acid chain. This latter thus
undergoes a deprotection processing of the protecting
t-butyloxycarbonyl groups by the various solvents, by means of
successive washings. Then, after introduction under pressure of the
solvent, the resin in suspension undergoes an agitation by bubbling
with nitrogen under weak pressure.
According to another characteristic of the invention, all of the
openings of the chemical reactor 2 are provided with control valves
12 assuring successively or simultaneously the control of the
inlets and the outlets of the reactor and being able totally to
isolate this latter from its peripheral context, the opening 30
situated at the lower extremity of the chemical reactor 2 being
connected, as shown in FIGS. 7 and 8, on the one hand, to a waste
valve 131 and, and on the other hand, to a bubbling valve 130 with
the aid of a T-shaped connector 11".
Each chemical reactor 2, associated with each volumetric solvent
proportioner 1, thus forms an assembly which is comprised as an
independent entity. It is therefore possible to use one or several
reactors 2, independently from one another, so as partially to
isolate one or several during the course of the synthesis, to
modulate the quantity of peptides produced by each one of the
reactors 2, and, consequently, to be able to synthesize
simultaneously variable quantities of peptides of different
sequences.
According to FIG. 4, the injection device 27 of the solvent under
pressure has at its upper part a coupling connection 31, and is
composed of a hollow cylinder 32, into which is introduced an open,
ribbed cylinder 33, of slightly lesser diameter and having on its
lower base an enlarged frustoconical portion 34 permitting a radial
and laminar distribution of the liquid flux injected into the
chemcial reactors 2.
The angle of the truncated cone 34 is preferably comprised between
50.degree. and 70.degree. and the height h of the injection device
27 inserted into the neck of the opening 26 is provided such that
the liquid injected comes in contact of the internal wall of the
chemical reactor 2 as well as the internal wall of the openings 28
and 29.
The height h of the injection device inserted into the neck of the
opening 26 is preferably about 10 mm shorter than the length of the
said neck. The diameter of the injection device 27 is less than the
diameter of the neck of the opening 26 of the chemical reactor 2 by
about 0.5 mm to 1.5 mm, such that the meniscus is formed between
the wall of the glass and the lower extremity of the ribbed
cylinder 33. Thus, the injected liquid recovers all the resin
particles which may adhere to the internal wall of the chemical
reactor 2 and simultaneously washes the openings 28 and 29 of the
chemical reactor 2.
According to FIG. 5, each distribution cylinder 3 is provided with
a concentric distribution channel 35, the extremities 36 and 37 of
which form the inlet and the outlet of the distributor, the
distribution being effected radially by means of channesl 38
disposed on different levels.
According to an embodiment, the channels 38 are advantageously
eight in number and on two levels, namely four channels per level,
and are disposed at 90.degree. with respect to one another.
According to another characteristic of the invention, as shown in
FIGS. 7 and 8, the number of distribution cylinders 3 is
advantageously eight, namely a general distribution cylinder 3A for
the solvents, a general distribution cylinder 3B for nitrogen, a
distribution cylinder 3C for low pressure nitrogen toward the
opening 16 of the volumetric proportioner 1 and for evacuation of
air and nitrogen from the same opening 16, a distribution cylinder
3D for low pressure nitrogen toward the opening 29 of the chemical
reactors 2 and for the evacuation of air and nitrogen from the same
opening 29, a general distribution cylinder 3E for evacuation of
air and nitrogen vapors toward a hood 44, a general distribution
cylinder 3F for evacuation of the waste, a general distribution
cylinder 3G for very low pressure nitrogen toward the opening 30 of
the chemical reactors 2 and a distribution cylinder for nitrogen
for the evacuation of the chemical reactor 2 (see FIGS. 7 and
8).
According to FIG. 6, the solvent selector 4 is traversed by a
concentric distribution channel 39, one extremity 40 of which is
connected by tubes 10 and connectors 11, 11' to one of the
distribution cylinders 3 and the other extremity 41 of which is
connected by tubes 10 and connectors 11, 11' to a waste recovery
device 42, and the various solvents being supplied to the
distribution channel 39 by means of channels 43 having openings
controlled by valves 120 and each disposed on a different level
according to the repeating interval of a helix. The solvent
selector 4 is designed such that it eliminates any risk of
contamination of the chemical reactors 2, momentarily isolated by
segments of parasitic fluid. Specifically, at the time of the
injection into the volumetric proportioners 1 of the most
aggressive solvent, in this case trifluoroacetic acid, this latter
contaminates on its passage the small injection channels 43 of the
other solvents. But the channels 43 each being disposed on a
different level according to the repeating interval of a helix so
as to be able to sequence the following solvents in a very precise
order, from the most aggressive to the least aggressive starting
from the base of the solvent selector 4, they are well cleansed by
a reflux motion of the liquid toward the waste recovery device 42.
There is thus assured a complete rinsing of the solvent selector
4.
The distribution cylinders 3 as well as the solvent selector 4 are
advantageously realized in PTFE.
The invention similarly has as an object a process for production
of synthetic peptides by use of the peptide multisynthesizer shown
in FIG. 1, the process consisting in realizing simultaneously and
independently the synthesis of a variable number of peptides in
different quantities, of identical or different sequences,
according to a common synthesis technique comprising, for each
cycle, either starting from an amino acid-resin couple that has
been introduced into each chemical reactor 2, or such a couple to
be realized for each volumetric solvent proportioner 1-chemical
reactor 2 assembly, then deprotecting the N-.alpha. amino group of
each amino acid by eliminating the t-butyloxycarbonyl protecting
group by acidolysis and neutralizing the amino group obtained in
the form of a salt, coupling the second protected amino acid to the
resin, deprotecting its N-.alpha. amino group and repeating this
cycle for each peptide to be produced, with identical or different
sequence, a number of times equal to the number of amino acids to
be fixed in the resin. This number of amino acids could therefore
be different from one chemical reactor 2 to the other. The first
peptide obtained will be that comprising the least amino acids and
the last obtained will be that comprising the most, the duration of
production of each peptide being proportional to the number of
cycles required, and thus to the number of amino acids to be fixed
in the resin.
The first step of each cycle of the synthesis technique consists,
either in introducing a resin/amino acid couple through the opening
28 of each chemical reactor 2, or generating it with the aid of the
multisynthesizer. For the generation of the resin/amino acid
couples, the following steps, for each volumetric solvent
proportioner 1-chemical reactor 2 assembly, are effected under the
control of the programmable automaton 7 or the microcomputer:
manual introduction of resin through the opening 28;
injection of a solvent under pressure of nitrogen, namely
dichloromethane, through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for an agitation by bubbling of about 1 min. so as to wash and to
inflate the resin by agitation of the solvent and the resin;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the solvent through the opening 30;
repetition of these three latter operations until obtention of a
suitable resin;
manual introduction of the amino acid protecting group
t-butyloxycarbonyl through the opening 28;
injection of a solvent under pressure of nitrogen, namely
dichloromethane, through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for about 4 hours for an agitation by bubbling with manual
introduction with several repetitions of coupling and/or activating
agents through the opening 28;
introduction of nitrogen under pressure through the opening 26, so
as to evacuate the solvent through the opening 30;
injection of a solvent under pressure of nitrogen; namely
dichloromethane, through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for about one minute for an agitation by bubbling so as to wash the
resin-amino acid couple;
introduction of nitrogen under pressure through the opening 29, so
as to evacuate the solvent through the opening 30;
repetition of these three letter operations until obtention of a
suitable resin-amino acid couple.
There will thus follow, for each resin-amino acid couple obtained,
the deprotection of the N-amino group, by elimination of the
protecting group t-butyloxycarbonyl by acidolysis according to the
following steps, affected under the control of the programmable
automaton 7 or the microcomputer for each volumetric solvent
proportioner 1-chemical reactor 2 assembly;
injection under pressure of nitrogen through the opening 26 of a
mixture of 35% dichloromethane and 65% trifluoroacetic acid;
introduction of very low pressure nitrogen through the opening 30
for about one minute, for an agitation by bubbling;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the dichloromethane-trifluoroacetic acid mixture
through the opening 30;
injection of the mixture of dichloromethane and trifluoroacetic
acid under pressure of nitrogen in equal proportions through the
opening 26;
introduction for about 13 minutes of very low pressure nitrogen
through the opening 30 for an agitation by bubbling;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the dichloromethane-trifluoroacetic acid mixture
through the opening 30;
injection of a solvent under pressure of nitrogen, namely
dichloromethane, through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for about one minute for an agitation by bubbling so as to wash the
deprotected amino acid-resin couple;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the solvent through the opening 30;
repetition of these three latter operations;
injection of a solvent under pressure of nitrogen, namely
dimethylformamide through the opening 26, so as to eliminate the
remaining trifluoroacetic acid;
introduction of very low pressure nitrogen through the opening 30
for several seconds, for an agitation by bubbling;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the solvent through the opening 30;
injection of a solvent, namely dichloromethane, through the opening
26;
introduction of very low pressure nitrogen through the opening 30
for about one minute for an agitation by bubbling;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the solvent through the opening 30;
injection of a solvent, namely dimethylformamide through the
opening 26;
introduction of very low pressure nitrogen through the opening 30
for about one min. for an agitation by bubbling;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the solvent through the opening 30;
repetition of this latter operation;
For the neutralization of the amino group obtained in the salt
form, the following steps, for each volumetric solvent proportioner
1-chemical reactor 2 assembly are effected under the control of the
programmable automaton 7 or the microcomputer:
injection under pressure of nitrogen of a mixture of 10%
diisopropylethylamine and 90% dichloromethane or dimethylformamide
through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for about one min. for an agitation by bubbling;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the diisopropylethylaminedichloromethane mixture or
the diisopropylethylaminedimethylformamide mixture through the
opening 30;
repetition of this latter operation;
injection of a solvent under pressure of nitrogen, namely
dichloromethane or dimethylformamide, through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for about one minute for an agitation by bubbling;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the solvent through the opening 30;
repeating three times this latter operation for the washing of the
resin-amino acid couple;
manual withdrawal through the opening 28 of a sample of the
resin-amino acid couple for verification of the deprotection
reaction with the aid of a test with ninhydrin.
Finally, for coupling the second protected amino acid onto the
obtained resin-amino acic couple, the following steps, for each
volumetric solvent proportioner 1-chemical reactor 2 assembly are
realized under the control of the programmable automaton 7 or the
microcomputer:
manual introduction of the protected amino acid, activated or
non-activated, through the opening 28;
injection of a solvent under pressure of nitrogen, namely
dichloromethane or dimethylformamide, through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for a duration comprised between 10 and 60 minutes for an agitation
by bubbling with manual introduction of coupling and/or activating
agents through the opening 28;
manual withdrawal through the opening 28 of a sample of the
obtained resin-amino acid assembly and test with ninhydrin;
either introduction of nitrogen under pressure through the opening
26 so as to evacuate the solvent through the opening 30, if the
test effected at the time of the preceding operation is negative,
or repeating of the operations starting from the neutralization of
the amino group obtained in the salt form, if the test effected at
the time of the said preceding operation is positive;
injection of a solvent under pressure of nitrogen, namely
dichloromethane, through the opening 26;
introduction of very low pressure nitrogen through the opening 30
for about one minute for agitation by bubbling so as to wash the
obtained resin-amino acid assembly;
introduction of nitrogen under pressure through the opening 26 so
as to evacuate the solvent through the opening 30;
repeating three times this latter operation until obtention of a
suitable assembly.
Throughout the cycle, the pressure of nitrogen necessary for
injection of the solvent is comprised between 300 and 400 mbar,
preferably between 340 and 360 mbar, and the pressure of nitrogen
for the agitation by bubbling is comprised between 10 and 70 mbar,
preferably between 20 and 35 mbar. This very low pressure is
maintained constant within .+-.1/10 mbar thanks to the center 5 for
adjustment and regulation of nitrogen. The volume of solvent to be
injected is comprised between 15 and 25 ml/gram of resin,
preferably 20 ml.
The programmable automaton 7 or microcomputer controls each
physical element of the multisynthesizer, so as to cause it to
execute the necessary functions throughout the course of each cycle
of the synthesis technique. If it is a case of a programmable
automaton, its microprocessor could advantageously be of the 8032
type from Intel Corp., the program memory of 4K octets ROM and the
assembly program language MCS 51.
The programmable automaton or microcomputer thus controls the
chronology of the various operations of each cycle, as well as each
operation itself.
The principal operations for each cycle are the measuring of the
solvent volumes, the injection of solvent into each chemical
reactor 2, the agitation by bubbling and the evacuating of each
chemical reactor 2.
According to a characteristic of the invention, the measuring of
the solvent volumes to be injected into each chemical reactor 2
through the opening 26 is effected under the control of the
programmable automaton or microcomputer 7 in the following
manner:
opening of the valve 120 for admission of the selected solvent and
120' for admission of the selected nitrogen;
opening of the admission valve 121 of the opening 16 of the
volumetric solvent proportioner 1;
opening of the valve 122 for exposing the distribution cylinder 3C
to the ambient;
injection of a solvent under pressure of nitrogen through the
opening 16 of the volumetric proportioner 1 until a detection of
the level by the strip 25 of optoelectronic detectors;
closing of the admission valve 121 of the opening 16 of the
volumetric solvent proportioner 1;
adjustment of the pre-established volume for each volumetric
proportioner 1-chemical reactor 2 assembly;
closing the admission valves 120 and 120' for the solvent-nitrogen
pair selected. According to another characteristic of the
invention, the injection of solvent into each chemical reactor 2 is
effected under the control of the programmable automaton 7 or
microcomputer in the following manner:
opening of the valve 123 for admission of nitrogen from the
distribution cylinder 3C so as to pressurize, through the opening
16 of the volumetric proportioner 1, the solvent contained in the
principal cylinder 13 and the measuring column 14 of the volumetric
proportioner 1;
placing in underpressure the chemcial reactor 2 by opening the
expansion valve 125 of the opening 29 through the distribution
cylinder 3D;
opening of the admission valve 126 of the opening 26;
placing in underpressure for about 5 seconds the volumetric
proportioner 1 by opening the valve 122 for exposing the
distribution cylinder 3C to the ambient.
According to another characteristic of the invention, the agitation
by bubbling in each chemical reactor 2 is effected under the
control of the programmable automaton 7 or microcomputer in the
following manner:
placing the reactor in underpressure by opening the expansion valve
125 of the opening 29 through the distribution cylinder 3D;
delay of 5 seconds for relaxation of the chemical reactor 2;
opening general valves 127, 127' for distribution of very low
pressure nitrogen above the distribution cylinder 3G;
opening the nitrogen admission valve 130 of the opening 30;
opening the valve 128 controlling the overpressure impulse for a
half second;
closing the valve 128;
distribution of nitrogen at very low controlled pressure for an
agitation by bubbling through the opening 30;
closing the nitrogen admission valve 130 of the opening 30;
delay of 200 ms prior to the beginning of evacuation.
According to another characteristic of the invention, the
evacuation of each chemical reactor 2 is effected under the control
of the programmable automaton or microcomputer 7 in the following
manner:
opening the valve 126' of the opening 26 for admission of nitrogen
from the distribution cylinder 3H;
opening the waste valve 124 of the distribution cylinder 3F;
opening the solvent evacuation valve 131 of the opening 30;
placing the reactor in underpressure by opening the waste valve 125
of the opening 29 through the distribution cylinder 3D;
delay of 5 seconds for relaxation of the chemical reactor 2.
The operations accompanying each cylinder are the rinsing of the
solvent selector 4 and the refilling of the solvent container 6.
The rinsing of the solvent selector 4 of the residual segments of
preceding solvent is effected under the control of the programmable
automaton 7 or microcomputer in the following manner:
opening the nitrogen admission valve 123 of the distribution
cylinder 3C;
opening the waste valve 129 of the solvent selector 4;
opening of the admission valve 121 of the opening 16.
The refilling of the solvent containers, is effected under the
control of the programmable automaton 7 or microcomputer in the
following manner:
closing the low pressure valve 132 of the distribution cylinder
3B;
opening the valve 133 for exposing the distribution cylinder 3B to
the ambient;
opening the valve 120' of the solvent container under
consideration;
manual refilling of the containers 6;
closing the valve 120' of the solvent container under
consideration;
closing the valve 133 for exposing the distribution cylinder 3B to
the ambient;
opening the low pressure valve 132 of the distribution cylinder
3B.
Finally, the programmable automaton or the microcomputer similarly
controls the liquid crystal display, the print-out, the injection
of solvent for the compensation of evaporation during long periods
of agitation by bubbling, as well as the alarm and safety
devices.
It will be understood that the inventionis not limited to the
embodiments described and shown in the accompanying drawings.
Modifications remain possible, particularly from the point of view
of the construction of the various elements, or by substitution of
equivalent techniques, without departing whatsoever from the scope
of protection of the invention.
* * * * *